Regeneration of craniofacial tissue provides one means to restore function to a disease- and injury-free state. Biochemical factors have successfully been used to differentiate stem cells to many cell types. However, little progress has been made in being able to spatially pattern tissue. Recent evidence indicates the ability to use bioelectric signaling as an alternative method to direct stem cell differentiation. Due to advances in specialized tools, it is now possible to spatially and temporally control bioelectric stimulation. The hypothesis of this project is that membrane potential (Vmem) is an instructive differentiation signal capable of inducing specific molecular pathways depending on the precise Vmem. Inducing tissue regeneration with Vmem stimulation could profoundly impact oral and maxillofacial defect treatment by for the first time enabling spatial patterning of hard and soft tissues. The goal of this project is to develop a system to screen modulation of Vmem for the ability to induce stem cell differentiation to a specific cell type and to study the mechanism behind Vmem regulation. The focus will be on the differentiation of human dental pulp stem cells (hDPSCs) to osteoblasts and adipocytes, two important craniofacial cell types. The first aim of this project is to develop a screen to identify beneficial Vmem for differentiation to particular cll types. The screen will require reproducible induction of Vmem and analysis of differentiation. Electrophysiological recordings from hDPSCs will be used to correlate media ion concentrations with induced membrane potentials. Currently available differentiation marker fluorescent protein (FP) reporter constructs will be validated as a means to monitor hDPSC differentiation. As a proof of principle, FP expression in hDPSCs cultured in decreased biochemical induction factors known to affect differentiation will be observed. It is expected that changes in FP expression will correlate with the effects on differentiation and provided similar resolution of monitoring differentiation as other forms of analysis, such as qPCR, but with the added benefit of being nondestructive. In aim 2, the screen will be used to identify membrane potentials beneficial and instructive to osteogenic and adipogenic differentiation. Hyperpolarizing and depolarizing membrane potentials will be induced and changes in FP expression will be used to identify effects on differentiation in media with or without biochemical induction factors. Changes in differentiation molecular pathways will be investigated to identify Vmem signal transduction mechanisms. The outcome of this aim will provide evidence of the utility of using bioelectric signaling to induce differentiation of specific cell types. This work will enable future developmen of patterned tissue regeneration for the purpose of craniofacial reconstruction and other clinical applications. PUBLIC HEALTH RELEVANCE: Despite much progress in stem cell and tissue regeneration research, little progress has been made in developing complicated multi-cell type tissues as would be required for many oral and craniofacial applications. This project will develop a screen to allow high throughput analysis of Vmem induced effects as well as expand fundamental knowledge of the role of membrane potential in directing stem cell fate. The outcome of the study will provide a new tool for regeneration with improved spatial control of tissue patterning, which would have a broad impact on craniofacial reconstruction.